PROJECT SUMMARY/ABSTRACT HIV-exposed, uninfected (HEU) infants experience significantly higher rates of morbidity and mortality due to respiratory syncytial virus (RSV) infection compared to HIV-unexposed (HUU) infants. Natural killer (NK) cells and antigen-presenting cells (APCs) are innate immune cells that play a critical role in controlling RSV infection. We have previously identified abnormalities in NK cells and APCs from HEU infants, but it is not clear whether these abnormalities explain the increased severity of RSV disease observed in this population. This research seeks to address that knowledge gap and will also investigate which in utero exposures are responsible for immune dysfunction in HEU infants. This research is relevant to the mission of the NICHD because it will advance the understanding of immune cross-talk between the pregnant woman and fetus by studying the impact of maternal HIV infection on neonatal innate immune function. The central hypothesis of this proposal is that in utero exposure to the inflammatory environment associated with maternal HIV infection induces DNA methylation changes in innate immune cells that alter NK cell and APC function, and ultimately impair the response to RSV infection. This hypothesis will be tested through the following specific aims: 1) To compare the innate immune response to RSV infection between HEU and HUU infants using an in vitro model of human respiratory infection; 2) To determine the effect of exposure to an environment enriched in inflammatory cytokines on neonatal innate immune cell function; 3) To identify differences in DNA methylation and RNA expression in NK cells and APCs between HEU and HUU infants and determine the effect of in vitro cytokine exposure on the epigenetic and transcriptomic profile of these cells. Aim 1 will be investigated using an innovative respiratory epithelial and endothelial co-culture system along with HEU and HUU cord or peripheral blood mononuclear cells. In Aim 2, HUU NK cells and APCs will be incubated with inflammatory cytokines to simulate HEU infants? in utero conditions. Aim 3 will generate the first description of the epigenome and transcriptome of HEU compared with HUU infants, using robust techniques including DNA methylation arrays and single cell RNA sequencing. This approach is innovative because it: 1) allows the first investigation of the impact of HEU immune dysregulation on RSV pathogenesis, and 2) may identify the mechanism for immune dysregulation in HEU infants. This project is significant because it has potential to improve health outcomes for the more than 1 million HEU infants born each year. Complimentary to the proposed research plan, a five-year mentored career development training plan has been devised that incorporates didactic learning in genomic data analysis and hands-on training in virology and immunology laboratory skills. The candidate is co-mentored by internationally recognized experts in the fields of virology, immunology, and genomics/bioinformatics. The candidate?s long- term career goal is to become an independent investigator studying the immune effects of HIV exposure.